Role Of Ras Signaling Research Articles

Role of RAS signaling in ovarian cancer.

The RAS family of proteins is among the most frequently mutated genes in human malignancies. In ovarian cancer (OC), the most lethal gynecological malignancy, RAS, especially KRAS mutational status at codons 12, 13, and 61, ranges from 6-65% spanning different histo-types. Normally RAS regulates several signaling pathways involved in a myriad of cellular signaling cascades mediating numerous cellular processes like cell proliferation, differentiation, invasion, and death. Aberrant activation of RAS leads to uncontrolled induction of several downstream signaling pathways such as RAF-1/MAPK (mitogen-activated protein kinase), PI3K phosphoinositide-3 kinase (PI3K)/AKT, RalGEFs, Rac/Rho, BRAF (v-Raf murine sarcoma viral oncogene homolog B), MEK1 (mitogen-activated protein kinase kinase 1), ERK (extracellular signal-regulated kinase), PKB (protein kinase B) and PKC (protein kinase C) involved in cell proliferation as well as maintenance pathways thereby driving tumorigenesis and cancer cell propagation. KRAS mutation is also known to be a biomarker for poor outcome and chemoresistance in OC. As a malignancy with several histotypes showing varying histopathological characteristics, we focus on reviewing recent literature showcasing the involvement of oncogenic RAS in mediating carcinogenesis and chemoresistance in OC and its subtypes.

Correlation between Ubiquitin E3 Ligases (SIAHs) and Heat Shock Protein 90 in Breast Cancer Patients.

Background:Breast cancer is a heterogeneous disease and differences in the expression levels of the ER, PR, and HER2 the triplet of established biomarkers used for clinical decision-making have been reported among breast cancer patients. Furthermore, resistance to anti-estrogen and anti-HER2 therapies emerges in a considerable rate of breast cancer patients, and novel drug therapies are required. Several anomalous signaling pathways have been known in breast cancer have been known; heat shock protein 90 (HSP90) is one of the most plenty proteins in breast cells. The family of ubiquitin ligases such as SIAH1 and SIAH2 is known to specifically target misfolded proteins to the proteasome; also, they have been illustrated to play a role in RAS signaling and as an essential downstream signaling component required for EGFR/HER2 in breast cancer.Methods:The expression of SIAH2, HSP90, and HER2 was assessed by quantitative Real-Time PCR in 85 invasive ductal carcinoma breast tumor samples at Uludag University Hospital in Turkey during the years 2018–2019, and its association with the clinicopathologic variables of patients was evaluated.Results:HSP90, SIAH1, and SIAH2 were significantly (P=0.0271, P=0.022, and P=0.0311) upregulated tumor tissue of patients with breast cancer. Moreover, this study observed a significant association between the high expression of SIAH2/HSP90 with ER status, high expression of HSP90 with Recurrence/Metastasis, and high expression of SIAH2 with Ki-67 proliferation index.Conclusion:The HSP90 and SIAH2 expressions play a significant role in breast cancer development by combining the experimental and clinical data obtained from the literature.

Characterization of bone homeostasis in individuals affected by cardio-facio-cutaneous syndrome.

Cardio-facio-cutaneous syndrome (CFCS) is a rare disorder characterized by distinctive craniofacial appearance, cardiac, neurologic, cutaneous, and musculoskeletal abnormalities. It is due to heterozygous mutations in BRAF, MAP2K1, MAP2K2, and KRAS genes, belonging to the RAS/MAPK pathway. The role of RAS signaling in bone homeostasis is highly recognized, but data on bone mineral density (BMD) in CFCS are lacking. In the present study we evaluated bone parameters, serum and urinary bone metabolites in 14 individuals with a molecularly confirmed diagnosis of CFCS. Bone assessment was performed through dual X-ray absorptiometry (DXA); height-adjusted results were compared to age- and sex-matched controls. Blood and urinary bone metabolites were also analyzed and compared to the reference range. Despite vitamin D supplementation and almost normal bone metabolism biomarkers, CFCS patients showed significantly decreased absolute values of DXA-assessed subtotal and lumbar BMD (p ≤ 0.05), compared to controls. BMD z-scores and t-scores (respectively collected for children and adults) were below the reference range in CFCS, while normal in healthy controls. These findings confirmed a reduction in BMD in CFCS and highlighted the importance of monitoring bone health in these affected individuals.

Abstract A05: RAS signaling promotes ERMS cell viability via sustaining TAZ expression and protein stability

Abstract Rhabdomyosarcoma (RMS) is a pediatric cancer characterized by skeletal muscle histogenesis, with the embryonal histologic variant (ERMS) being the most common. Mutations of the RAS pathway are found in 40-60% of ERMS patient specimens, and as in other human cancers the RAS protein is currently not druggable, and inhibition of downstream RAS effector pathways fails due to compensating signaling. Thus, there is an urgent need to understand the dysregulated signaling in RAS-driven ERMS (rERMS). Hippo signaling was originally identified for its role in controlling organ size and tissue homeostasis, but it is now known to also have a critical role in cancer initiation and progression. In ERMS patients, higher YAP1 gene expression correlates with a poor prognosis, and in vitro and in vivo studies using ERMS cell lines and mouse models also show that YAP1 supports ERMS cell and tumor growth. Whether and how the YAP1 paralog WWTR1 (TAZ) contributes to rERMS tumorigenesis remains understudied. Our initial studies examining ERK suppression (genetic inhibition via shRNA or pharmacologic inhibition via trametinib) showed that this slowed rERMS cell growth and proliferation, and induced rERMS cell differentiation. However, we also found CTGF and CYR61 (downstream targets of YAP1/TAZ) significantly repressed upon trametinib treatment. While YAP1 appeared unaffected, trametinib treatment significantly suppressed TAZ at both the mRNA and protein levels in a dose-dependent manner. Suppression of TAZ with shRNA decreased rERMS cell growth and proliferation, suggesting that sustained TAZ expression is crucial for rERMS cell viability. Interestingly, there was no change in the activity of MST and LATS. Our next focus will be on the mechanism of ERK-mediated TAZ expression, with a potential role for glycogen synthase kinase 3 beta as a mediator of TAZ degradation. In summary, these studies identify a new role for RAS signaling in promoting rERMS cell survival via TAZ expression. Citation Format: Liz Yi-Tzu Lin, Corinne Linardic. RAS signaling promotes ERMS cell viability via sustaining TAZ expression and protein stability [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A05.

LGALS1 acts as a pro-survival molecule in AML

The galectin LGALS1 is a glycan binding protein that regulates intracellular (e.g. signal transduction) and extracellular processes (e.g. immunity, leukocyte mobilization) that support cell survival. The protein is best known for its role in RAS signaling. LGALS1 is important in acute lymphoblastic leukemia but its role in acute myeloid leukemia is not well defined. We previously found suppression of LGALS1 in AML cell lines OCI-AML3 and THP-1 sensitized both cell lines to BCL2 inhibitor ABT-737. In this study, we used an in vivo murine OCI-AML3 xenograft model to test whether reduction expression of LGALS1 affects survival. Mice bearing the OCI-AML3 cells with LGALS1 shRNA survived significantly longer than mice with control OCI-AML3 cells. Gene expression profiling using RNASeq was performed using the control and LGALS1 shRNA of p53 WT OCI-AML3 and p53 mutant THP-1 cells. The data reveal distinct differences between the two cell lines in number of genes affected, in pathways associated with these genes, in expression of oncogenes, and in the transcription factors involved. The p53 pathway is prominent in OCI-AML3 cells. An examination of LGALS1 mRNA in an AML patient population reveals elevated LGALS1 mRNA is associated with shorter disease free survival and increased blasts in the BM. This data with the xenograft model data presented suggest LGALS1 may be important in the AML microenvironment. In summary, the data presented here suggest that a strategy targeting LGALS1 may benefit AML patients.

Ras Signaling Regulates Stem Cells and Amelogenesis in the Mouse Incisor

The role of Ras signaling during tooth development is poorly understood. Ras proteins—which are activated by many upstream pathways, including receptor tyrosine kinase cascades—signal through multiple effectors, such as the mitogen-activated protein kinase (MAPK) and PI3K pathways. Here, we utilized the mouse incisor as a model to study how the MAPK and PI3K pathways regulate dental epithelial stem cells and amelogenesis. The rodent incisor—which grows continuously throughout the life of the animal due to the presence of epithelial and mesenchymal stem cells—provides a model for the study of ectodermal organ renewal and regeneration. Utilizing models of Ras dysregulation as well as inhibitors of the MAPK and PI3K pathways, we found that MAPK and PI3K regulate dental epithelial stem cell activity, transit-amplifying cell proliferation, and enamel formation in the mouse incisor.

A review of craniofacial and dental findings of the RASopathies.

The RASopathies are a group of syndromes that have in common germline mutations in genes that encode components of the Ras/mitogen-activated protein kinase (MAPK) pathway and have been a focus of study to understand the role of this pathway in development and disease. These syndromes include Noonan syndrome (NS), Noonan syndrome with multiple lentigines (NSML or LEOPARD syndrome), neurofibromatosis type 1 (NF1), Costello syndrome (CS), cardio-facio-cutaneous (CFC) syndrome, neurofibromatosis type 1-like syndrome (NFLS or Legius syndrome) and capillary malformation-arteriovenous malformation syndrome (CM-AVM). These disorders affect multiple systems, including the craniofacial complex. Although the craniofacial features have been well described and can aid in clinical diagnosis, the dental phenotypes have not been analysed in detail for each of the RASopathies. In this review, we summarize the clinical features of the RASopathies, highlighting the reported craniofacial and dental findings. Review of the literature. Each of the RASopathies reviewed, caused by mutations in genes that encode different proteins in the Ras pathway, have unique and overlapping craniofacial and dental characteristics. Careful description of craniofacial and dental features of the RASopathies can provide information for dental clinicians treating these individuals and can also give insight into the role of Ras signalling in craniofacial development.

One-Pot N2C/C2C/N2N Ligation To Trap Weak Protein-Protein Interactions.

Weak transient protein-protein interactions (PPIs) play an essential role in cellular dynamics. However, it is challenging to obtain weak protein complexes owing to their short lifetime. Herein we present a general and facile method for trapping weak PPIs in an unbiased manner using proximity-induced ligations. To expand the chemical ligation spectrum, we developed novel N2N (N-terminus to N-terminus) and C2C (C-terminus to C-terminus) ligation approaches. By using N2C (N-terminus to C-terminus), N2N, and C2C ligations in one pot, the interacting proteins were linked. The weak Ypt1:GDI interaction drove C2C ligation with t1/2 of 4.8 min and near quantitative conversion. The Ypt1-GDI conjugate revealed that binding of Ypt1 G-domain causes opening of the lipid-binding site of GDI, which can accommodate one prenyl group, giving insights into Rab membrane recycling. Moreover, we used this strategy to trap the KRas homodimer, which plays an important role in Ras signaling.

Abstract 2457: Mechanisms of membrane binding of K-Ras4B farnesylated hypervariable region

Abstract K-Ras4B belongs to a family of small GTPases that regulates cell growth, differentiation and survival. Kras is frequently mutated in cancer. K-Ras4B modulates downstream signaling at different lipid microdomains. K-Ras4B association with the plasma membrane through its farnesylated and positively charged C-terminal hypervariable region (HVR) is critical to its oncogenic function. HVR farnesylation plays an important role in Ras signaling, but the structural mechanisms and consequences of farnesyl-induced membrane binding are poorly understood and the basis for Ras localization to certain parts of plasma membrane is unknown. Application of confocal microscopy, surface plasmon resonance, and molecular dynamics (MD) simulations revealed that K-Ras4B is distributed between rigid lipid rafts and disordered non-raft regions of the plasma membrane. Its membrane binding domain interaction with phospholipids is driven by membrane fluidity. The farnesyl group spontaneously inserts into the disordered lipid microdomains containing unsaturated phospholipids, while the rigid microdomains composed of tightly packed saturated lipids restrict the farnesyl group penetration. We speculate that the resulting farnesyl protrusion towards the cell interior allows oligomerization of K-Ras4B membrane binding domain in rigid microdomains. Unlike other Ras isoforms, K-Ras4B HVR contains a single farnesyl modification and positively charged polylysine sequence. The high positive charge not only modulates specific HVR binding to anionic phospholipids, but the farnesyl's membrane orientation. Phosphorylation of Ser181 prohibits spontaneous farnesyl membrane insertion. We speculate that if the farnesyl group is not inserted into the membrane, it may serve in cooperative binding of other K-Ras4B molecules, thus leading to dimerization. In this case, the protein binds the membrane using electrostatic interaction between its positively charged HVR and the negatively charged membrane phospholipids. In the case of disordered phospholipid domains, the farnesyl group burial in the phospholipids tends to hinder its interactions. The mechanism illuminates the roles of HVR modifications in K-Ras4B targeting to microdomains of the plasma membrane and suggests an additional function for HVR in regulation of Ras signaling. Elucidating the mechanism for K-Ras4B localization at different membrane microdomains is important for in-depth understanding of Ras-effector interactions mediating signaling pathways. Funded by Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract HHSN261200800001E. Citation Format: Hyunbum Jang, Sherwin J. Abraham, Tanmay S. Chavan, Ben Hitchinson, Lyuba Khavrutskii, Nadya I. Tarasova, Ruth Nussinov, Vadim Gaponenko. Mechanisms of membrane binding of K-Ras4B farnesylated hypervariable region. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2457. doi:10.1158/1538-7445.AM2015-2457

Abstract 3285: Importance of Ras signaling in EphB2-mediated ependymoma development

Abstract Ependymoma account for 6-12% of pediatric brain tumors and have a mean age of diagnosis of 4 years, a 5-year survival rate ranging from 39%-64%, and a progression free survival rate of 23-45%. General use of chemotherapeutics remains highly ineffective for this disease therefore standard of care consists of surgery followed by radiation with total gross resection (TGR) having the best outcome (60-89% 5-year overall survival for TGR vs. 21-46% 5-year overall survival for subtotal resection (STR)) [6]. Patients having had STR have an extremely high incidence of disease reoccurrence (43% to 72%) requiring additional surgeries and radiation with a median 2-year overall survival after relapse ranging from 20% to 49% [7]. These statistics highlight the need for better adjuvant therapies. In an effort to identify new targets for treating ependymoma we have developed a mouse model that relies on the intracranial implantation of mouse supratentorial (forebrain) embryonic neuronal stem cells (STeNSC) overexpressing the tyrosine kinase receptor ephrin B2 (EphB2). Tumors develop within 4-8 months post-implantation, require receptor activation and are consistently found to be histologically, ultrastructurally and molecularly similar to the human disease. Given the requirement for EphB2 activation in tumor development, it became necessary to identify the EphB2 initiated signaling pathways involved in this process. Among the many pathways initiated by EphB2 activation are those of the small GTPases of the Ras family (H-Ras, K-Ras, N-Ras) that regulate cellular processes including cell proliferation and transformation. Receptor activation is a multistep process involving receptor autophosphorylation of two critical tyrosines positions 604 and 610 within the juxtamembrane (Y604, Y610), receptor/ligand complex dimerization, release of juxtamembrane mediated kinase domain inhibition and finally kinase domain phosphorylation and activation. While EphB2-mediated activation of Ras involves the kinase domain of the receptor, Ras inhibition (RasGTP conversion to RasGDP) is achieved by activating the Ras GTPase activating protein p120RasGAP via its juxtamembrane. We took advantage of a previously identified EphB2 juxtamembrane mutation that inhibits p120RasGAP activation by converting the tyrosines at positions 604 and 610 to glutamic acids (Y604/610E) thus rendering Ras constitutively active. Ligand-mediated activation of this mutant resulted in an absence of p120RasGAP activation and an increase in Ras signaling (elevated levels of phosphorylated C-Raf, MEK1/2 and Erk1/2) in vitro as predicted. More importantly, overexpression of the EphB2(Y604/610E) protein in our model system showed no change in tumor penetrance (100% penetrance); however, there was a dramatic shortening of tumor latency from 207 days with EphB2 to 40 days with the Y604/610E mutation, supporting the role for Ras signaling in EphB2 mediated transformation. Citation Format: Robert A. Johnson, Phylip Chen, Robert Lyons, Samuel Priddy. Importance of Ras signaling in EphB2-mediated ependymoma development. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3285. doi:10.1158/1538-7445.AM2015-3285

A non-cell-autonomous role for Ras signaling in C. elegans neuroblast delamination.

Receptor tyrosine kinase (RTK) signaling through Ras influences many aspects of normal cell behavior, including epithelial-to-mesenchymal transition, and aberrant signaling promotes both tumorigenesis and metastasis. Although many such effects are cell-autonomous, here we show a non-cell-autonomous role for RTK-Ras signaling in the delamination of a neuroblast from an epithelial organ. The C. elegans renal-like excretory organ is initially composed of three unicellular epithelial tubes, namely the canal, duct and G1 pore cells; however, the G1 cell later delaminates from the excretory system to become a neuroblast and is replaced by the G2 cell. G1 delamination and G2 intercalation involve cytoskeletal remodeling, interconversion of autocellular and intercellular junctions and migration over a luminal extracellular matrix, followed by G1 junction loss. LET-23/EGFR and SOS-1, an exchange factor for Ras, are required for G1 junction loss but not for initial cytoskeletal or junction remodeling. Surprisingly, expression of activated LET-60/Ras in the neighboring duct cell, but not in the G1 or G2 cells, is sufficient to rescue sos-1 delamination defects, revealing that Ras acts non-cell-autonomously to permit G1 delamination. We suggest that, similarly, oncogenic mutations in cells within a tumor might help create a microenvironment that is permissive for other cells to detach and ultimately metastasize.

Mobilization with granulocyte colony-stimulating factor blocks medullar erythropoiesis by depleting F4/80+VCAM1+CD169+ER-HR3+Ly6G+ erythroid island macrophages in the mouse

Similarly to other tissues, the bone marrow contains subsets of resident tissue macrophages, which are essential to maintain bone formation, functional hematopoietic stem cell (HSC) niches, and erythropoiesis. Pharmacologic doses of granulocyte colony-stimulating factor (G-CSF) mobilize HSC in part by interfering with the HSC niche-supportive function of BM resident macrophages. Because bone marrow macrophages are key to both maintenance of HSC within their niche and erythropoiesis, we investigated the effect of mobilizing doses of G-CSF on erythropoiesis in mice. We now report that G-CSF blocks medullar erythropoiesis by depleting the erythroid island macrophages we identified as co-expressing F4/80, vascular cell adhesion molecule-1, CD169, Ly-6G, and the ER-HR3 erythroid island macrophage antigen. Both broad macrophage depletion, achieved by injecting clodronate-loaded liposomes, and selective depletion of CD169(+) macrophages, also concomitantly depleted F4/80(+)VCAM-1(+)CD169(+)ER-HR3(+)Ly-6G(+) erythroid island macrophages and blocked erythropoiesis. This more precise phenotypic definition of erythroid island macrophages will enable studies on their biology and function in normal settings and on diseases associated with anemia. Finally, this study further illustrates that macrophages are a potent relay of innate immunity and inflammation on bone, hematopoietic, and erythropoietic maintenance. Agents that affect these macrophages, such as G-CSF, are likely to affect these three processes concomitantly.

Global analysis of induced transcription factors and cofactors identifies Tfdp2 as an essential coregulator during terminal erythropoiesis

Key transcriptional regulators of terminal erythropoiesis, such as GATA-binding factor 1 (GATA1) and T-cell acute lymphocytic leukemia protein 1 (TAL1), have been well characterized, but transcription factors and cofactors and their expression modulations have not yet been explored on a global scale. Here, we use global gene expression analysis to identify 28 transcription factors and 19 transcriptional cofactors induced during terminal erythroid differentiation whose promoters are enriched for binding by GATA1 and TAL1. Utilizing protein-protein interaction databases to identify cofactors for each transcription factor, we pinpoint several co-induced pairs, of which E2f2 and its cofactor transcription factor Dp-2 (Tfdp2) were the most highly induced. TFDP2 is a critical cofactor required for proper cell cycle control and gene expression. GATA1 and TAL1 are bound to the regulatory regions of Tfdp2 and upregulate its expression and knockdown of Tfdp2 results in significantly reduced rates of proliferation as well as reduced upregulation of many erythroid-important genes. Loss of Tfdp2 also globally inhibits the normal downregulation of many E2F2 target genes, including those that regulate the cell cycle, causing cells to accumulate in S phase and resulting in increased erythrocyte size. Our findings highlight the importance of TFDP2 in coupling the erythroid cell cycle with terminal differentiation and validate this study as a resource for future work on elucidating the role of diverse transcription factors and coregulators in erythropoiesis.

Dual function of MyD88 in inflammation and oncogenesis

Inflammation is emerging as a new hallmark of cancer, and the toll-like receptor and interleukin-1 receptor adaptor molecule MyD88 has been linked to tumorigenesis. The purpose of this review is to give a brief overview of the latest advances in understanding the complexity of MyD88 implication in tumorigenesis. MyD88 is shown to play a protumorigenic role through two mechanisms. First, it activates the nuclear factor kappa-light-chain-enhancer of activated B cells signaling pathway in the hematopoietic compartment and in tumor cells, inducing an inflammatory environment favorable to carcinogenesis. Second, it plays a cell-autonomous role in Ras signaling and transformation, independently of its role in inflammatory signaling. MyD88 mediates the optimal activation of the Ras/extracellular signal-regulated kinase (ERK) pathway by binding to ERK and protecting it from dephosphorylation. This optimal activation of the Ras pathway is essential for the expression of important DNA repair enzymes, allowing cancer cells to efficiently repair damaged DNA. MyD88 is also shown in certain cases to play an antitumoral role through modulation of the immune response These findings present a new dual function model for MyD88 implication in carcinogenesis making it a potential therapeutic target in cancer.

Mammalian Adenylyl Cyclase-associated Protein 1 (CAP1) Regulates Cofilin Function, the Actin Cytoskeleton, and Cell Adhesion

CAP (adenylyl cyclase-associated protein) was first identified in yeast as a protein that regulates both the actin cytoskeleton and the Ras/cAMP pathway. Although the role in Ras signaling does not extend beyond yeast, evidence supports that CAP regulates the actin cytoskeleton in all eukaryotes including mammals. In vitro actin polymerization assays show that both mammalian and yeast CAP homologues facilitate cofilin-driven actin filament turnover. We generated HeLa cells with stable CAP1 knockdown using RNA interference. Depletion of CAP1 led to larger cell size and remarkably developed lamellipodia as well as accumulation of filamentous actin (F-actin). Moreover, we found that CAP1 depletion also led to changes in cofilin phosphorylation and localization as well as activation of focal adhesion kinase (FAK) and enhanced cell spreading. CAP1 forms complexes with the adhesion molecules FAK and Talin, which likely underlie the cell adhesion phenotypes through inside-out activation of integrin signaling. CAP1-depleted HeLa cells also had substantially elevated cell motility as well as invasion through Matrigel. In summary, in addition to generating in vitro and in vivo evidence further establishing the role of mammalian CAP1 in actin dynamics, we identified a novel cellular function for CAP1 in regulating cell adhesion.

Abstract 5524: Library screen to rapidly determine activity against normal and mutant bone marrow progenitor cells.

Abstract Aberrant signal transduction plays a central role in the pathogenesis of myelodysplastic syndrome/ myeloproliferative neoplasm (MDS/MPN), as indicated by the high prevalence of mutations that activate Ras signaling. Yet despite the key role of Ras signaling in hematopoietic neoplasms, there are currently no signal transduction inhibitors with established efficacy. This necessitates a screen of inhibitors that may potentially reveal novel therapeutic strategies and inform efforts to treat neoplasms driven by hyperactive Ras signaling. Unfractionated bone marrow cells harvested from Mx1-Cre, KrasD12 (n= 10) and wildtype (WT) mice (n=18) were utilized in the screening of 94 different inhibitors. A disease relevant, homogenous population of PreGM cells identified as Lineage lo/- Sca1- c-kit+ CD34+ FcγRII/III- CD105- CD150- was purified from bone marrow using flow cytometry. The PreGM cells were then sorted into 96 well plates containing various inhibitors at set concentrations ranging from 1E-7× (5E-7ug/ml) to 1× (5ug/ml). DMSO and cytotoxic agents (Cytarabine, Adriamycin, Gemcitabine) served as negative and positive controls respectively. The freshly sorted PreGM cells were exposed to inhibitors for 3 days under standard culture conditions. At the end of that period, cell growth was quantified using the IN Cell Analyzer 2000 and dose response curves constructed for WT and mutant cells. WT and mutant IC50s for each compound were calculated using the ‘drc’ package from the R Project for Statistical Computing. The 94 drug candidates tested in this screen included cytotoxic agents, tyrosine kinase inhibitors, PI3K family inhibitors, mitotic kinase inhibitors, epigenetic modifiers, hedgehog signaling inhibitors, and others. Candidates were screened for preferential activity against Mx1-Cre, KrasD12 cells. However, none of the compounds tested were found to demonstrate preferential inhibitory activity against Mx1-Cre, KrasD12 cells when compared to WT cells. Furthermore, the IC50s calculated for mutant and WT cells were not significantly different across all inhibitors tested. The majority of compounds tested were either FDA approved drugs or agents used in recent clinical trials. Hyperactive Kras signaling is a major underlying etiology for MDS/MPN, and thus we designed our study to test the efficacy of these candidates against mutant Kras cells. Our results indicate that mutant cells have similar drug sensitivities as normal cells over a broad range of mechanistic approaches. This leads us to believe that "synthetic" lethal opportunities may not be the approach of choice for treating Kras driven neoplasms. However, these findings have directed our efforts towards compounds that target upstream mediators of Kras signaling and future endeavors are currently underway to use the assay system to assess the activity of a highly curated library of kinase inhibitors on mutant and WT proliferation. Citation Format: Wan Xing Hong, Jon Akutagawa, Steven Chen, Michelle Arkin, Benjamin S. Braun. Library screen to rapidly determine activity against normal and mutant bone marrow progenitor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5524. doi:10.1158/1538-7445.AM2013-5524

Dermatological phenotype in Costello syndrome: consequences of Ras dysregulation in development

The RASopathies are a class of human genetic syndromes caused by germline mutations in genes that encode protein components of the Ras/mitogen-activated protein kinase (MAPK) pathway. Costello syndrome (CS) is a RASopathy caused by mutations in the HRAS gene, a key regulator of signal transduction. To quantify the specific cutaneous phenotype observed in 46 individuals with Costello syndrome with confirmed HRAS mutations. This was a cross-sectional study. Dermatological surveys were designed by the authors and were completed by parents of mutation-positive individuals with CS at the Costello Syndrome Family Network (CSFN) conferences in 2007 and 2009. Dermatological examinations were performed by the authors at the CSFN conferences. Cutaneous papillomas were reported in 33 of the 46 (72%) participants, with age of onset ranging from infancy to 22years. Individuals with CS are more likely than patients with cardiofaciocutaneous syndrome (CFC) to present with cutaneous papillomas (72% vs. 5%, P<0·001) and palmoplantar keratoderma (76% vs. 36%, P<0·001). Individuals with CS are less likely than individuals with CFC to present with sparse or absent eyebrows (9% vs. 90%, P<0·001) or keratosis pilaris (33% vs. 80%, P=0·001). This study also identified that loose, redundant skin on the hands and feet, 'stippled' dermatoglyphs (pachydermatoglyphia) on the fingertips (eight of 26, 31%) and acanthosis nigricans (17 of 46, 37%) are frequent features of CS. While there is significant phenotypic overlap among syndromes of the Ras/MAPK pathway, individuals with CS are more likely than individuals with CFC syndrome to present with cutaneous papillomas, palmoplantar keratoderma and full eyebrows, and are less likely to present with ulerythema ophryogenes, keratosis pilaris or multiple naevi. The dermatological features of CS, a Ras dysregulation syndrome, share many features with cutaneous paraneoplastic syndromes. This may provide further insight into the role of Ras signalling in cutaneous paraneoplastic syndromes.

Downregulation of tropomyosin‐1 in squamous cell carcinoma of esophagus, the role of Ras signaling and methylation

Tropomyosins (TMs) are a family of cytoskeletal proteins that bind to and stabilize actin microfilaments. Non-muscle cells express multiple isoforms of TMs including three high molecular weight (HMW) isoforms: TM1, TM2, and TM3. While reports have indicated downregulation of TMs in transformed cells and several human cancers, nevertheless, little is known about the underlying mechanism of TMs suppression. In present study the expression of HMW TMs was investigated in squamous cell carcinoma of esophagus (SCCE), relative to primary cell cultures of normal esophagus by western blotting and real-time RT-PCR. Our results showed that TM1, TM2, and TM3 were significantly downregulated in cell line of SCCE. Moreover, mRNA level of TPM1 and TPM2 were markedly decreased by 93% and 96%, in tumor cell line relative to esophagus normal epithelial cells. Therefore, downregulation of TMs could play an important role in tumorigenesis of esophageal cancer. To asses the mechanism of TM downregulation in esophageal cancer, the role of Ras dependent signaling and promoter hypermethylation were investigated. We found that inhibition of two Ras effectory downstream pathways; MEK/ERK and PI3K/Akt leads to significant increased expression of TM1 protein and both TPM1 and TPM2 mRNAs. In addition, methyltransferase inhibition significantly upregulated TM1, suggesting the prominent contribution of promoter hypermethylation in TM1 downregulation in esophageal cancer. These data indicate that downregulation of HMW TMs occurs basically in SCCE and the activation of MEK/ERK and PI3K/Akt pathways as well as the epigenetic mechanism of promoter hypermethylation play important role in TM1 suppression in SCCE.

A role for Ras signaling in modulating mammalian aging by the GH/IGF1 axis

The discovery that mutations in single genes can modulate aging was not only fascinating but it provided researchers with animal models with which to study aging in cohorts of different aging rates [1-2]. According to the GenAge database [3], at the time of writing, genetic manipulations in 68 genes have been shown to affect lifespan in mice. Among mouse genes in which mutations extend lifespan, a number are part of the growth hormone/insulin-like growth factor 1 (GH/IGF1) axis. As such, it is widely acknowledged that decreased GH/IGF1signaling in mice can extend lifespan, while increased GH levels may accelerate aging [4-5]. Although the GH/IGF1axis is one of the major pathways regulating aging in mammals, its precise downstream mechanisms remain a subject of debate and scrutiny. In this year's March issue of AGING, Borras et al. show that mice deficient in Rasgrf1, a guanine nucleotide–releasing factor for Ras, exhibit a significant increase (~20%) in average and maximum lifespan. This increase in lifespan was independent of cancer mortality and was accompanied by a retardation in age–related functional and physiological decline [6]. Ras homologues have been associated with lifespan modulation in yeast [7], so it seems that Ras signaling is another example of an evolutionary conserved pathway modulating aging. Rasgrf1 is unique among Ras family members, however, as it is only expressed in a few specific tissues, in particular in pancreatic β–cells and in some regions of the brain. Thus far, Rasgrf1 has been primarily associated with learning and memory [6,8-9]. Because of its expression in the brain, Rasgrf1 has been shown to regulate the synthesis and release of GH, and previous studies showed that Rasgrf1 deficient mice have lower levels of GH [9-10]. In line with these previous studies, and given that GH impacts on IGF1 levels, Rasgrf1 deficient mice showed lower IGF1 levels [6]. Decreased GH/IGF1 signaling results in stunted growth and reduced adult body size, which has been associated with life-extension in numerous models [11]. Borras et al. and other authors also observed that Rasgrf1deficient mice are smaller than controls [6,9-10]. Therefore, it is likely that decreased GH/IGF1 signaling is the mechanism by which Rasgrf1 impacts on aging. Changes in GH/IGH1 have also been hypothesized to be related to the mechanisms of caloric restriction [4-5], which is in line with the observation by Borras et al., that Rasgrf1 deficient mice have metabolic profiles similar to mice under caloric restriction. Because Rasgrf1 mutants have altered GH/IGF1 signaling, I had predicted that they would be long-lived [12]. Moreover, it was reported that bi-maternal mice are long-lived and, because Rasgrf1is an imprinted gene expressed from the paternal allele in neonates, it is possible that bi-maternal mice are long-lived due to Rasgrf1 effects [12-13]. Although conducted in different labs and strains, bi–maternal mice had a similar increase in average lifespan (28%), though a more modest increase in maximum lifespan (~5%). Rasgrf1 deficient mice are therefore another model to study the modulation of aging by the GH/IGF1 axis. Interestingly, mice expressing Rasgrf1from both alleles have increased body size and higher IGF1 levels [9]. Although the lifespan of these animals is unknown, they could be seen as a complementary model to the Rasgrf1 knock-outs. Consequently, since they are available with both increased GH (biallelic Rasgrf1 mice) and decreased GH levels (Rasgrf1 knock outs), the different Rasgrf1 mutant mice may be an excellent new model to study aging and its modulation by the GH/IGF1 axis.

RAS signaling pathway mutations and hypertrophic cardiomyopathy: getting into and out of the thick of it

In this issue of the JCI, Wu et al. and Marin et al. describe two new mouse models of inherited disorders of the RAS/MAPK signal transduction pathway that display hypertrophic cardiomyopathy (HCM); the model from the former paper was from a gain-of-function Raf1 mutation, and the model from the latter paper was from a protein tyrosine phosphatase, non-receptor type 11 (Ptpn11) mutated allele encoding Shp2 with impaired catalytic function. The two groups show that HCM arises from increased signaling through Erk1/2 and the mTor complex 1, respectively, and that those cardiac issues can be prevented or reversed with small-molecule therapies inhibiting the appropriate pathway. Aside from being the first studies of treatment for Noonan syndrome and related disorders in a mammalian system, these papers provide important insights into the role of RAS signaling in cardiac hypertrophy and suggest the complexity in developing meaningful therapy for individuals with these RASopathies.